Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor.
Endothelium-dependent vasorelaxation in large vessels is mainly attributed to Nomega-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and Nomega-nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-ArgNO2-L-Dbu-NH2 2TFA (L-ArgNO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-ArgNO2-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-ArgNO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H2O2 in mediating vasodilatation.
Capettini
L S A
LS
Department of Physiology and Biophysics, ICB, Federal University of Minas Gerais. Av. Antônio Carlos, 6627, Pampulha 31270-901, Belo Horizonte, MG, Brazil.
Cortes
S F
SF
Gomes
M A
MA
Silva
G A B
GA
Pesquero
J L
JL
Lopes
M J
MJ
Teixeira
M M
MM
Lemos
V S
VS
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000201:enzymology*; D001685:Biological Factors / Q000378:metabolism*; D002374:Catalase / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D045462:Endothelium-Dependent Relaxing Factors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006834:Hydrazines / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D019335:Nitroarginine / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014664:Vasodilation / Q000187:drug effects; D014665:Vasodilator Agents / Q000494:pharmacology
D001685:Biological Factors; D045462:Endothelium-Dependent Relaxing Factors; D004791:Enzyme Inhibitors; D006834:Hydrazines; D007093:Imidazoles; D020030:Nitric Oxide Donors; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D014665:Vasodilator Agents; C067410:endothelium-dependent hyperpolarization factor; D009599:Nitroprusside; D019335:Nitroarginine; C098186:1-(2-trifluoromethylphenyl)imidazole; D009569:Nitric Oxide; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; D006861:Hydrogen Peroxide; D002374:Catalase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; C496321:Nos1 protein, mouse; C496315:Nos3 protein, mouse; D000109:Acetylcholine; D019331:NG-Nitroarginine Methyl Ester